Nucleotide sequence analysis of DNA is one of the basic techniques in molecular biology.
As methods for determining DNA sequences, there have been known two fundamental methods, i.e., the Maxam-Gilbert method (chemical degradation method) [Methods Enzymology, 65, 499-560 (1980)] and the Sanger method (dideoxy chain termination method) [Proc. Natl. Acad. Sci., USA, 74, 5463-5467 (1977)]. Among these, the dideoxy chain termination method is simpler and enables more quick sequencing in comparison with the chemical degradation method, and therefore it has been the mainstream of the DNA sequencing methods.
The basic principle of the dideoxy chain termination method is as follows.
First, a single-stranded DNA containing a DNA fragment of which nucleotide sequence is to be determined is prepared, and used as a template for the replication step. Then, a primer is annealed to a region of the DNA in the proximity of the inserted region of the DNA fragment, and a DNA complementary to the single-stranded DNA is synthesized with an enzyme called Klenow fragment. This synthesis is performed in the presence of the four kinds of naturally occurring 2'-deoxyribonucleotides and 2',3'-dideoxynucleotides labeled with radioactive isotopes, fluorescent dyes and the like and corresponding to the four kinds of nucleotides as chain terminators (terminators). The 2',3'-dideoxynucleotides are 2'-deoxyribonucleotides of which 3'-OH groups are replaced by H groups, and they may be the substrate for the Klenow fragment like the 2'-deoxyribonucleotides, but the DNA chain extension is terminated when the 2',3'-dideoxynucleotides are incorporated. As a result, various DNA chains having common 5'-ends but having various chain lengths are synthesized.
That is, the above reaction is performed by using the 2',3'-dideoxynucleotide compounds together with 2'-deoxyribonucleotides for all of the bases, adenine (A), guanine (G), cytosine (C), and thymine (T), and the products are subjected to electrophoresis. This enables reading of the order of the nucleotide sequence based on the sizes of the DNA fragments.
However, under the recent circumstance where total DNA sequence or even whole genomic genes of humans, animals and plants are determined as in, for example, the so-called human genome project, there have been pointed out problems of the above dideoxy chain termination method, for example, complexity of the operation procedure (for preparation of template single-stranded DNA and the like), difficulty of shortening the process time and the like.
As the method for determining DNA sequences, the method where products amplified by the polymerase chain reaction (PCR) method [Randall K. Saiki et al. Science, 239:487-491 (1988)] are sequenced without cloning them [the direct sequencing method, Corinne Wong et al. Nature, 330:384-386 (1988)] is also a useful method. This method does not require the library construction and screening, and is a quick method capable of simultaneously obtaining sequence information of many samples.
However, the above direct sequencing method suffers from two major problems. One is that primers and 2'-deoxyribonucleoside 5'-triphosphates (2'-dNTPs) not incorporated remain in the reaction system, and they inhibit the sequencing reaction. Therefore, in conventional methods, such primers and 2'-dNTPs must be removed from PCR products before sequencing. There are many methods for purification of PCR products such as purification by electrophoresis, ethanol precipitation, gel filtration, HPLC purification and the like [see, for example, Dorit R. L et al. Current Protocols in Molecular Biology, Vol. 11, John Wiley and Sons, New York, 15.2.1-15.2.11 (1991)]. However, these methods are complicated without exception.
The second problem is quick renaturation of PCR products. When the PCR products are renatured into a double-stranded DNA, they are no longer single-stranded templates, and annealing between primers and single-stranded templates is inhibited. As methods for minimizing the renaturation, there have been reported, for example, quenching after denaturation, biotilation of one primer and absorption of PCR products onto streptavidin-coated articles, use of exonuclease, asymmetric PCR and the like. They are disclosed in, for example, Barbara Bachmann et al., Nucleic Acid Res., 18:1309 1990. However, most of these methods are time-consuming and very laborious.
DNA sequencing methods by chain elongation utilizing RNA polymerases have been researched as one of the means which solve these problems. Among such DNA sequencing methods utilizing RNA polymerases, there has been known a method utilizing four kinds of naturally occurring nucleotides and labeled 3'-deoxynucleotides having radioisotopes such as .sup.32 P as the label and corresponding to the four kinds of nucleotides as terminators [Biochemistry, 24, 5716-5723 (1985)].
However, because these labeled terminators contain radioactive isotopes as the label, they are not preferred from the viewpoints of safety for human bodies and waste disposal. Therefore, it is expected to use terminators having fluorescent labels in stead of radioisotopes.
As for the labeled terminators used for the dideoxy chain termination method mentioned above, there have been various reports, for example, the report of Sanger et al. [J. Mol. Biol., 143, 161-178 (1980)], the report of Smith et al. [Nucleic Acids Res., 13, 2399-2412 (1985)], the report of Plober et al. [Science, 238, 336-341 (1987)], the report of Connel et al. [BioTechniques, 5, 342-348 (1987)], the report of Lee et al. [Nucleic Acids Res., 20, 2471-2483 (1992)], PCT International Application Unexamined publication in Japanese (KOHYO) No. (Hei) 5-502371/1993, Japanese Patent Publication (KOKOKU) No. (Hei) 7-121239/1995 and the like. However, all of the methods mentioned in these reports are those utilizing DNA polymerases, and therefore they use 2',3'-dideoxyribonucleotides as the terminators, and do not refer fluorescence-labeled 3'-deoxyribonucleotides at all.
Japanese Patent Publication No. (Hei) 8-5908/1996 mentions various labeled 2',3'-dideoxyribonucleotides, labeled 2'-deoxyribonucleotides, labeled 3'-deoxyribonucleotides and labeled ribonucleotides. However, as for the labeled 3'-deoxyribonucleotides, though they are disclosed as compounds falling within the scope of the general formula mentioned in the claims, they are not specifically exemplified as working examples and their synthesis is not verified. Therefore, they are considered still uncompleted. In addition, this patent document does not mention examples of actual use of the nucleotide derivatives for DNA sequencing at all.
On the other hand, it has been known that the incorporation of 2'-deoxyribonucleotides by DNA polymerase during DNA synthesis shows fluctuation depending on a base group each nucleotide has. Similar tendency is expected for labeled nucleotide derivatives, and fluctuation of the incorporation by DNA polymerase depending on differences between the labels is also expected. In spite of this, the above Japanese Patent Publication No. (Hei) 8-5908/1996 did not evaluate the incorporation of the disclosed derivatives by DNA polymerase at all. Labeled 3'-deoxyribonucleotides were of course not evaluated, since such compounds themselves were not synthesized.
The situation explained above is for the DNA sequencing methods using DNA polymerases, and it has not been known yet what kind of tendency would be observed for DNA sequencing methods using RNA polymerases. That is, there has not been any substantial knowledge about a difference in the incorporation efficiency among ribonucleotides resulting from a base group difference and a difference in the incorporation efficiency of labeled 3'-deoxyribonucleotides as well as the influence of these differences on DNA sequencing methods. In the fields requiring such an exquisite technique as DNA sequencing, fluorescence-labeled compounds and fluorescence-labeled 3'-deoxyribonucleotides are used for the dideoxy chain termination method which imposes the severe requirement that the materials must not inhibit the activity of RNA polymerase. Therefore, it is extremely difficult to estimate fluorescence-labeled terminators useful for the chain termination method using RNA polymerases from known labeled terminators such as those mentioned above based on structural correlation between DNA polymerases and RNA polymerases and the like.
Under these circumstances explained above, the present inventors previously developed various fluorescence-labeled 3'-deoxyribonucleotide derivatives useful as the terminators for the method of determining nucleotide sequences of nucleic acids utilizing RNA polymerases, and filed a patent application therefor (Japanese Patent Application No. (Hei) 8-227904/1996).
Thereafter, those fluorescence-labeled 3'-deoxyribonucleotide derivatives were actually used in methods for determining nucleotide sequences of nucleic acids utilizing RNA polymerases, and as a result nucleotide sequences of template DNA could be determined. However, even though the reason has not been clear, those fluorescence-labeled 3'-deoxyribonucleotide derivatives could not be incorporated by RNA polymerases with an efficiency sufficient for practical use in methods for determining DNA sequences. Therefore, in order to enable practical use of the DNA sequence determination methods utilizing RNA polymerases, it has been desired to provide fluorescence-labeled 3'-deoxyribonucleotide derivatives with improved incorporation rates by RNA polymerases.
Accordingly, an object of the present invention is to provide 3'-deoxyribonucleotide derivatives with improved incorporation rates by RNA polymerases, which are useful as terminators in the DNA sequence determination methods utilizing RNA polymerases.